New Tools Add Precision to Live-Cell Fluorescence

Dennis Doherty, Prior Scientific Inc.

Researchers using live-cell fluorescence often have to examine samples repeatedly to observe changes over time. Viewing multiple samples in three dimensions as a time-lapse movie is common in labs such as the Nikon Imaging Center at Harvard Medical School in Boston. But before automated imaging platforms that combine a stage, focus motor, filter wheel and shutter were developed, this type of high-resolution fluorescence was nearly impossible.

Prior Scientific’s filter wheel system fits onto the excitation and emission ports of a microscope and can be run from a single controller or from a keypad.

Creating a time-lapse movie enables the researcher to document details of a process and to quantify pertinent data. It involves a series of steps, snapping images at different positions, with different filters and at different intervals, and integrating them to provide a complete set of clear, in-focus, detailed three-dimensional images.

Each step sounds fairly simple, yet the process would be impossible without the precise movements of a stage and focus, and the speed of a filter wheel and shutter. According to Nikon Imaging Center lab director Jennifer Waters Shuler, there is no way to position samples accurately by hand, let alone manipulate them quickly enough. Prior Scientific Inc. of Rockland, Mass., has supplied the center with automated filter wheels and shutters.

Prior’s high-speed shutters are used alone or in conjunction with filter wheels. Minimizing the exposure time the specimen has to the many forms of light used in fluorescence imaging is critical to ensuring a long usable sample life.

A typical routine for such applications as transference between two cells would be to first find the X, Y and Z areas of interest on the prepared sample. If, for example, there are three areas with groups of cells of interest, each area will require four fields of view in X and Y and eight focal planes (Z) to cover completely. This requires navigation to 32 planes. Filters must be used in each focal plane to provide the single-color images that will be integrated into a multicolor image. Typically, a red, a green and a blue image are taken. That’s three images and six shutter moves per focal plane, for a total of 96 images.

Multiple moves

To capture these, the camera is triggered 96 times, but there are also 32 position-translation moves at submicron resolution and 96 filter moves, and the shutter will open and close 192 times. Perhaps it’s determined that images must be taken in five-minute intervals over one hour. That’s 13 sets, or 416 submicron-resolution repeatable translation moves, 1248 filter wheel positions and images captured, and 2496 shutter moves. And that is only for the first of three areas of interest.

Software can control and synchronize the movements of the hardware, and can stitch, place and deconvolve the images into a series that can be played as a movie. Important are how accurate the intervals are in the different Z planes for deconvolution, and how accurately the stage moves so that the images in the X and Y planes fit together side by side and on top of one another. If the positioning is not accurate, the finished product will not be of high quality.

The speed of the data being processed also is important, because an image cannot be captured until the previous one is completed. And because hundreds of images are being taken at a high rate of speed, download speed and storage capacity are critical.

Clearly, today’s microscopy demands automated imaging platforms. Without continuing advances and developments in automation as well as in optics, cameras, computers and software, research would be painfully slow and data collection far less accurate. Years ago, science drove the research, but now technology is the prerequisite.

Contact: Dennis Doherty, Eastern Regional Sales Manager, Prior Scientific Inc., Rockland, Mass.; +1 (800) 877-2234; fax: +1 (781) 878-8736; e-mail: ddoherty@prior.com.